NADH:ubiquinone oxidoreductase (complex I) is an energy-transducing membrane-bound protein complex that serves as a major entry point for electrons to the mitochondrial respiratory chain. The multisubunit enzyme complex is encoded by 43 separate genes in mammals. The mammalian enzyme shows extremely complex kinetic behavior, in all forms that retain natural electron transfer pathways. The complex kinetic behavior suggests that two distinct slowly equilibrating forms of the enzyme exist. The active (A) form is catalytically capable to reduce ubiquinone, whereas, the de-active (D) form is not able to transfer electrons to ubiquinone in the membrane. The equilibrium between these two forms is under a refined control by a number of regulatory factors (i.e., pH, divalent cations, temperature, the ubiquinone/ubisemiquinone/ubiquinol ratio). This research will primarily be done in Russia as an extension of NIH grant R01 GM61606. The proposed collaborative work is to extend studies of the regulatory characteristics of the enzyme at the molecular mechanistic level by using a combination of biochemical and molecular approaches. The studies will be done in physiologically relevant systems such as intact hearts and mitochondria, as well as, the kinetic and biochemical approaches in the foreign Co-P.l.'s laboratory. Two major aspects of the ND transition are to be investigated. One is to elucidate the molecular mechanisms of the slow interconversion between the A and D forms involved in the red/ox-dependent D to A transition; and the extremely temperature-dependent A to D transition. Natural ligands and modifiers which affect the rate and equilibrium of the ND transition in vitro will be identified. The second aim focusses on the complex I A/D transition in intact mitochondria in relation to the experimental model of ischemia/reperfusion in Langendorff-pertused rat hearts. The focus of these studies is to determine the possible physiological importance of the A/D transition of complex I as it responds to alterations in cellular metabolism. The catalytic activity of complex I is important for cellular physiology and a number of diseases (diabetes, cardiomyopathy) have been associated with defects in complex I. The research to be undertaken will shed light on the modulation of enzyme activity of this important respiratory complex.